Not applicable.
This invention is directed to methods for producing combinatorial chemistry libraries containing hydroxylamines and hydroxylamine derivatives, including hydroxamic acid derivatives, hydroxylurea derivatives, and hydroxylsulfonamide derivatives. This invention is further directed to synthesis of combinatorial chemistry libraries of hydroxylamines and hydroxylamine derivatives, including hydroxamic acid derivatives, hydroxylurea derivatives, and hydroxylsulfonamide derivatives, using solid-phase techniques. This invention is still further directed to the libraries of hydroxylamines and hydroxylamine derivatives, including hydroxamic acid derivatives, hydroxylurea derivatives, and hydroxylsulfonamide derivatives, produced by the solid-phase synthetic method disclosed. This invention is still further directed to utilizing the libraries of hydroxylamines and hydroxylamine derivatives (including hydroxamic acid derivatives, hydroxylurea derivatives, and hydroxylsulfonamide derivatives) to identify and select compounds which bind to, inhibit, or otherwise affect enzymes, receptors, or other biological molecules implicated in disease processes (including disease-related metalloproteases). The hydroxylamines and hydroxylamine derivatives (including hydroxamic acid derivatives, hydroxylurea derivatives, and hydroxylsulfonamide derivatives) thus selected have potential therapeutic value.
The techniques of combinatorial chemistry have been increasingly exploited in the process of drug discovery. Combinatorial chemistry allows for the synthesis of a wide range of compounds with varied molecular characteristics. Combinatorial synthetic techniques enable the synthesis of hundreds to millions of distinct chemical compounds in the same amount of time required to synthesize one or a few compounds by classical synthetic methods. Subjecting these compounds to high-throughput screening allows thousands of compounds to be rapidly tested for desired activity, again saving time expense and effort in the laboratory.
Chemical combinatorial libraries are diverse collections of molecular compounds. Gordon et al. (1995) Acc. Chem. Res. 29:144-154. These compounds are formed using a multistep synthetic route, wherein a series of different chemical modules can be inserted at any particular step in the route. By performing the synthetic route multiple times in parallel, each possible permutation of the chemical modules can be constructed. The result is the rapid synthesis of hundreds, thousands, or even millions of different structures within a chemical class.
For several reasons, the initial work in combinatorial library construction focused on peptide synthesis. Furka et al. (1991) Int. J. Peptide Protein Res. 37:487-493; Houghton et al. (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; Fodor et al. (1991) Science 251:767. The rapid synthesis of discrete chemical entities is enhanced where the need to purify synthetic intermediates is minimized or eliminated; synthesis on a solid support serves this function. Construction of peptides on a solid support is well-known and well-documented. Obtaining a large number of structurally diverse molecules through combinatorial synthesis is furthered where many different chemical modules are readily available; hundreds of natural and unnatural amino acid modules are commercially available. Finally, many peptides are biologically active, making them interesting as a class to the pharmaceutical industry.
The scope of combinatorial chemistry libraries has recently been expanded beyond peptide synthesis. Polycarbamate and N-substituted glycine libraries have been synthesized in an attempt to produce libraries containing chemical entities that are, similar to peptides in structure, but possess enhanced proteolytic stability, absorption and pharmacokinetic properties. Cho et al. (1993) Science 261:1303-1305; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9367-9371. Furthermore, benzodiazepine, pyrrolidine, and diketopiperazine libraries have been synthesized, expanding combinatorial chemistry to include heterocyclic entities. Bunin et al. (1992) J. Am. Chem. Soc. 114:10997-10998; Murpy et al. (1995) J. Am. Chem. Soc. 117:7029-7030; and Gordon et al. (1995) Biorg. Medicinal Chem. Lett. 5:47-50.
Hydroxylamines and their derivatives, including hydroxamic acids, hydroxyl ureas, and hydroxyl sulfonamides, have been the subject of much research focused on their properties as metalloprotease inhibitors. Izquierdo-Martin et al. (1992) J. Am. Chem. Soc. 114:325-331; and Cushman et al. (1981) Chapter 5 xe2x80x9cSpecific Inhibitors of Zinc Metallopeptidasesxe2x80x9d in Topics in Molecular Pharmacology (Burgen and Roberts, eds.). Metalloproteases are believed, to be involved in the development of arthritis, tumor angiogenesis, retinopathy, and many other disease processes.
U.S. Pat. No. 5,268,384 discloses hydroxamates and hydroxyl ureas used to treat inhibit angiogenesis by inhibiting matrix metalloproteases. Among metalloproteases disclosed as targets of inhibitors are collagenases, including human skin fibroblast collagenase and purulent human sputum collagenase; gelatinases, including human skin fibroblast gelatinase and purulent human sputum gelatinase; and stromelysin. Disclosed disorders amenable to treatment by matrix metalloprotease (MMP) inhibitors include ocular pathologies such as diabetic retinopathy and neovascular glaucoma; cancer, including Kaposi""s sarcoma, glioblastoma, and angiosarcoma; immune system disorders such as rheumatoid arthritis; and skin disorders such as psoriasis.
Patent publication WO 96/26918 discloses hydroxamates for inhibiting MMPs. The publication also discusses the inhibition of the production or the action of the cytokine tumor necrosis factor (TNF) by hydroxamic acid MMP inhibitors. See also, Mohler et al. Nature 370:218-220 (1994); Gearing et al., Nature 370:555-557 (1994); and McGeehan et al., Nature 370:558-561 (1994). These MMP inhibitors are described as useful for treating inflammatory, infectious, immunological or malignant diseases due to their effect on TNF. Among the specific diseases described are septic shock, hemodynamic shock, malaria, meningitis, fibrotic disease, cachexia, autoimmune diseases, and graft rejection.
Patent publication WO 96/25156 discloses hydroxamates for inhibiting matrix metalloproteases. The publication also discusses inhibition of production or processing of transforming growth factor alpha (TGF-xcex1) by MMP inhibitors, and describes potential applications of the MMP inhibitors in treating inflammation; wound healing, including scar and keloid formation; diabetic retinopathy; neovascular glaucoma; atherosclerosis; vascular adhesions; systemic lupus erythrematosus; various carcinomas; and other diseases amenable to treatment by modulating production or processing of TGF-xcex1.
U.S. Pat. No. 5,552,419 discloses aryl sulfonamido-substituted hydroxamic acids. The compounds are described as inhibitors of stromelysin, gelatinase and/or collagenase. Disorders described as amenable to treatment by the hydroxamic acid derivatives are osteoarthritis and rheumatoid arthritis; tissue ulceration; periodontal disease; bone diseases, including Paget""s disease and osteoporosis; HIV infection; and tumor metastasis, tumor progression or tumor invasion.
Patent publication EP 423943 describes the use of inhibitors of certain matrix metalloproteases, such as collagenases, gelatinases, and stromelysins, as useful for treatment of demyelinating diseases such as multiple sclerosis and other scleroses; demyelinating peripheral neuropathies; acute disseminated encephalomyelitis; and other neural disorders.
Other hydroxamic acid-based metalloprotease inhibitors are described in the following patent publications: U.S. Pat. Nos. 4,599,361 and 5,256,657; European patent publications EP 236872, EP 274453, EP 489577, EP 489579, EP 497192, EP 574758; and international PCT applications WO 90/05716, WO 90/05719, WO 91/02716, WO 92/13831, WO 92/22523, WO 93/09090, WO 93/09097, WO 93/20047, WO 93/24449, WO 93/24475, WO 94/02446, WO 94/02447, WO 94/21612, WO 94/25434, and WO 94/25435.
Many synthetic routes to produce hydroxylamines have been developed and are well-known in the art (see the above-cited publications for representative examples). These methods are limited by the necessity of preparing one compound at a time. Solid-phase synthesis of an immobilized hydroxamate is mentioned in patent application WO 96/26918; however, the method used in the application is limited to the Ugi reaction described. See also, Strocker et al. Tet. Lett. 37:1149-1152 (1996); Keating et al., J. Am. Chem. Soc. 118:2574-2583 (1996); and Tempest et al. Angew. Chem. Int. Ed. Engl. 35:640-642 (1995), and references therein.
The invention disclosed herein provides a method for combinatorial synthesis of hydroxylamines and hydroxylamine derivatives, enabling synthesis of a much greater variety of compounds in a relatively short amount of time.
All references, publications and patents mentioned herein are hereby incorporated herein in their entirety.
The present invention provides a method of synthesizing a combinatorial library of hydroxylamines and hydroxylamine derivatives on a solid support, where the first step of the method is the nucleophilic addition of an alkoxyamine to an appropriate solid support. The alkyl group forming the alkoxy portion of the alkoxyamine may be a protecting group, or may be intended to remain a part of the final compounds. The solid support bound alkoxyamine is then derivatized. Following derivatization, the alkoxyamine derivatives are optionally deprotected and cleaved from the solid support.
In another embodiment, the combinatorial libraries are synthesized by adding an O-alkoxy-protected hydroxylamine-linker intermediate comprising an O-protected alkoxyamine and a linker group to a solid support bearing an amine group, derivatizing the alkoxyamine, and then optionally deprotecting the alkoxyamine derivatives and cleaving them from the solid support.
In another embodiment, the method is used to synthesize hydroxylamine and hydroxylamine derivatives selected from the group consisting of hydroxylamines, hydroxamic acids, hydroxyl ureas, and hydroxyl sulfonamides.
The invention also encompasses libraries of the compounds synthesized by the methods described. These libraries are composed of a plurality of distinct compounds where the classes of compounds include, but are not limited to, hydroxylamines and hydroxylamine derivatives, including hydroxamic acids, hydroxylureas, and hydroxylsulfonamide derivatives. The libraries preferably contain at least about 40, 50, 80, 100, 500, 1000, 5000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 distinct compounds, depending on the reactions used for derivatization at each step and the degree of diversity desired in the library.
In yet another embodiment, the method is used to synthesize compounds of the formulas: 
wherein the R groups are independently selected from the group consisting of H, alkyl, heteroalkyl, aryl, heteroaryl, and heterocyclic moieties as defined herein, as well as amino acid side chains (both naturally and non-naturally occurring as defined herein); m, n, and x are integers independently selected from 0 to 12; and y is an integer selected from 0 to 30. The R groups can be attached to asymmetric carbon atoms in either the R-configuration or the S-configuration; additionally, all stereoisomeric and diasteromeric variations of the compounds and substituents are included in the invention. All protected derivatives of the compounds and all salts of the compounds are also included in the invention.
These libraries include compounds of the form:
L3xe2x80x94L2xe2x80x94L1xe2x80x94NHOH,
where
L3 is selected from the group consisting of 
L2 is selected from the group consisting of 
and L1 is selected from the group consisting of 
where the wavy bonds indicate the points of attachment to the rest of the molecule.
These libraries also include compounds of the form
L12xe2x80x94S(xe2x95x90O)2xe2x80x94L11xe2x80x94NHOH,
where
L12 is selected from the group consisting of 
and L11 is selected from the group consisting of 
These libraries also include compounds of the form
L22xe2x80x94S(xe2x95x90O)2xe2x80x94L21xe2x80x94NHOH
where L22 is selected from the group consisting of 
and L21 is selected from the group consisting of 
where Z is
These libraries also include compounds of the form: 
where R1 is selected from the group consisting of 
and L is selected from the group consisting of 
These libraries also include compounds of the form:
L2xe2x80x94L1xe2x80x94NHOH
where L2 is selected from the group consisting of xe2x80x94H, xe2x80x94C(xe2x95x90O)xe2x80x94CH3, xe2x80x94C(xe2x95x90O)xe2x80x94OCH3, and xe2x80x94C(xe2x95x90O)xe2x80x94CH2xe2x80x94C(xe2x95x90O)xe2x80x94OH; and L1 is selected from the group consisting of 
The invention also encompasses O-protected hydroxylamine functionalized resins, prepared by displacing a leaving group on a solid support by adding an alkoxylamine nucleophile with an alkoxy protecting group, resulting in a solid support bound alkoxyamine. These alkoxylamine nucleophiles can be O-trityl hydroxylamine, O-(t-butyldimethylsilyl) hydroxylamine, O-allyl hydroxylamine, O-benzyl hydroxylamine, O-(4-methoxybenzyl)hydroxylamine, O-(2,4-dimethoxybenzyl)hydroxylamine or O-(2-tetrahydropyranyl)hydroxylamine. The leaving group can be bromide, iodide, or mesylate.
The invention also encompasses compounds of the formula 
wherein b is an integer from 1 to 5, X is a leaving group selected from the group consisting of bromide, iodide, mesylate, tosylate, and p-nitrophenylsulfonate, and RESIN is any amine-bearing resin.
The invention also encompasses O-protected hydroxylamine functionalized resins, prepared by adding an O-protected hydroxylamine-linker intermediate to a solid support bearing an amine group, producing a solid support bound alkoxyamine.
The invention also encompasses an O-protected hydroxylamine-linker intermediate suitable for attachment to an amine-bearing resin. Such a compound is made up of an acid-labile linker group and an O-protected hydroxylamine.
The invention also encompasses compounds of the formula 
where b is an integer from 1 to 5, P1 is a protecting group selected from the group consisting of 2-tetrahydropyranyl, trityl, t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups, J2 is xe2x80x94H or -Fmoc, and J1 is xe2x80x94OH or xe2x80x94NH-RESIN, where RESIN is any solid or polymeric support.
The invention also encompasses derivatized hydroxymethylphenoxy resins and derivatized 2-methoxy-4-alkoxybenzyl alcohol resins, where the active hydroxyl group of the resin is replaced with a leaving group. This leaving group can be bromide, iodide, mesylate, tosylate, or p-nitrophenylsulfonate.
The invention also encompasses compounds of the formula: 
where b is an integer from 1 to 5, P1 is a protecting group selected from the group consisting of 2-tetrahydropyranyl, trityl, t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups, J2 is xe2x80x94H or -Fmoc, and J1 is xe2x80x94OH or xe2x80x94NH-RESIN, where RESIN is any solid or polymeric support.
The invention also encompasses compounds of the formulas: 
where b is an integer from 1 to 5, and P1 is a protecting group selected from the group consisting of 2-tetrahydropyranyl, trityl, t-butyldimethylsilyl, allyl, benzyl, 4-methoxybenzyl, and 2,4-dimethoxybenzyl protecting groups.
The invention also encompasses methods of use of the libraries synthesized by the combinatorial methods to screen for pharmacologically active compounds.